Synthesis and biological activity of certain ... - ACS Publications

J . Med. Chem. 1986,29, 268-278

268

Synthesis and Biological Activity of Certain Nucleoside and Nucleotide Derivatives of Pyrazofurin Charles R. Petrie 111, Ganapathi R. Revankar,* N. Kent Dalley, Rose D. George, Patricia A. McKernan, Robert L. Hamill,+ and Roland K. Robins Cancer Research Center, Department of Chemistry, Brigham Young University, Provo, Utah 84602, and The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206. Received May 8, 1985

A number of nucleoside and nucleotide derivatives of 4-hydroxy-3-~-~-ribofuranosylpyrazole-5-carboxamide (pyrazofurin, 1) were prepared and tested for their antiviral and cytostatic activity in cell culture. Treatment of 1 with benzyl bromide gave 4-0-benzylpyrazofurin (4). Methylation of 4 with CH2N2and subsequent removal of the benzyl group by catalytic hydrogenation provided 1-methylpyrazofurin (8). Direct methylation of 1 with CHJ furnished 4-0-methylpyrazofurin (6). Dehydration of the pentaacetylpyrazofurin (9) with phosgene furnished 4-acetoxy3-(2,3,5-tri-0-acetyl-~-~-ribofuranosyl)-l-acetylp~azole-5-carbonitrile (10). A similar dehydration of the precursor tetraacetyl derivative of 4 gave the corresponding carbonitrile, which on deprotection and subsequent treatment (13). Treatment of with hydroxylamine furnished 4-(benzyloxy)-3-~-~-ribofuranosylpyrazole-5-carboxamidoxime the tetraacetyl derivative of 4 with Lawesson’s reagent and subsequent deacetylation furnished a mixture of 4-(benzyloxy)-3-~-~-ribofuranosylpyrazole-5-thiocarboxamide (15) and the corresponding nitrile derivative (16). Phosphorylation of unprotected 4 with POCl, and subsequent debenzylation of the intermediate 17 gave pyrazofurin 5’-phosphate (18), which provided the first chemical synthesis of 18. Similar phosphorylation of 4 with POCl, and quenching the reaction mixture with either EtOH or MeOH, followed by debenzylation, furnished the 5’-0-(ethyl phosphate) (19b) and 5’-O-(dimethyl phosphate) (20b) derivatives of pyrazofurin. DCC-mediated cyclization of 17, followed by debenzylation, gave pyrazofurin 3’,5’-(cyclic)phosphate (21b). The NAD analogue 23b was also prepared by the treatment of 17 with an activated form of AMP in the presence of AgN03. The structural assignment of 7 , 8 , and 20a were made by single-crystal X-ray analysis, and along with pyrazofurin, their intramolecular hydrogen bond characteristics have been studied. All of these compounds were tested in Vero cell cultures against a spectrum of viruses. Compounds 18 and 23b were active a t concentrations very similar to pyrazofurin but are less toxic to the cells than pyrazofurin. Compounds 19b, 20b, and the 3’,5’-(cyclic)phosphate 21b are less active than 1. Compounds 18, 19b, 20b, and 23b also exhibited significant inhibitory effects on the growth of L1210 and P388 leukemias and Lewis lung carcinoma cells in vitro, whereas B16 melanoma cells were less sensitive to growth inhibition by these comDounds. Pvrazofurin derivatives modified a t the 1-,4-, or 5-position showed neither antiviral nor cytostatic actiGity in cell “culture.

The naturally occurring azolecarboxamide nucleoside antibiotic pyrazofurin (4-hydroxy-3-P-~-ribofuranosylpyrazole-5-carboxamide, pyrazomycin, 1) is one of three biologically active carbon-linked nucleosides isolated from the broth of a strain of Streptomyces candidus.’ Pyrazofurin is structurally related to the naturally occurring imidazole nucleoside bredinin (4-carbamoyl- l-P-D-ribofuranosylimidazolium-5-olate, 2),2as well as the synthetic triazole ribonucleoside ribavirin (1-0-D-ribofuranosyl1,2,4-triazole-3-carboxamide, 3),3 and shows significant

R

?,

mg/kg per d a y 9 . De Clercq and Torrence6 suggest that this unexpected toxicity is probably not associated with the structural features of the molecule responsible for the antiviral potency. However, the toxicity of pyrazofurin is such that it cannot readily be separated from its antiviral efficacy in a n i m a l ~ . ~ JIt~ has been presumed that the toxicity of pyrazofurin in animals m a y be due to its inhibition of orotidylate decarboxylase,11J2 an enzyme of the de novo pyrimidine biosynthetic pathway responsible for the transformation of orotidine 5’-monophosphate into uridine 5’-monophosphate. Pyrazofurin is similar to ribavirin (3) in that it is converted t o the 5’-triphosphate i n h u m a n blood cells13 and also converted to the 5’(1) Gerzon, K.; Williams, R. H.; Hoehn, M.; Gorman, M.; DeLong, D. C. 2nd Int. Congr. Heterocycl. Chem. 1969, Abstr. C-30,

HO

OH

HO OH

HO OH

1

2

3

pyrazofurin

bredinin

ribavirin

antiviral activity i n cell culture against a broad spectrum Pyrazofurin is particularly of DNA and RNA active against pox-, picorna-, toga-, myxo-, rhabdo-, and retroviruses in vitro and shows nearly as broad an antiviral ,~ is also highly efspectrum as r i b a ~ i r i n . ~ Pyrazofurin fective i n vitro against representative arena-, bunya-, and togavirus groups at concentrations 10-100 times less than those used with ribavirin.8 Although pyrazofurin has a high degree of selectivity in its antiviral effects and shows a rather broad safety margin i n cell culture, it exhibited a profound toxicity i n mice (50% lethality at a dose of 5 ‘The Lilly Research Laboratories, Eli Lilly and Company.

131. (2) Mizuno, K.; Tsujino, M.; Takada, M.; Hayashi, M.; Atsumi, K.; Asano, K.; Matsuda, T. J . Antibiot. 1974, 27, 775. (3) Witkowski, J. T.; Robins, R. K.; Sidwell, R. W.; Simon, L. N. J . Med. Chem. 1972,15, 1150. (4) Gutowski, G. E.; Sweeney, M. J.; DeLong, D.C.; Hamill, R. L.; Gerzon, K.; Dyke, R. W. Ann. N.Y. Acad. Sci. 1975,255,544. (5) Descamps, J.; De Clercq, E. ‘Current Chemotherapy”;Siegenthaler, W., Luthy, R., Ed; American Society for Microbiology: Washington, DC, 1978; p 354. (6) De Clercq, E.; Torrence, P. F. J. Carbohydr. Nucleosides Nucleotides 1978, 5, 187. (7) Shannon, W. M. Ann. N.Y. Acad. Sci. 1977,284, 472. (8) Canonico, P. G.; Jahrling, P. B.; Pannier, W. L. Antiuiral Res. 1983, 2, 331. (9) Streightoff, F. J.; Nelson, J. A.; Cline, J. C.; Gerzon, K.; Hoehn, M.; Williams, R. H.; Gorman, M.; DeLong, D. C. 9th Intersci. Conler. Antirnicrob. Agents Chemother. 1969, Abstr. 18. (10) DeLong, D. C.; Baker, L. D.; Gerzon, K.; Gutowski, G. E.; Williams, R. H.; Hamill, R. L. 7th Int. Congr. Chemother. 1971, A-5/35. (11) Ohnuma, T.; Roboz, J.; Shapiro, M. L.; Holland, J. F. Cancer Res. 1977, 37, 2043. (12) Cadman, E. C.; Dix, D. E.; Handschumacher,R. E. Cancer Res. 1978, 38, 682.

0022-2623/86/l829-0268$01.50/00 1986 American Chemical Society

Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2

Nucleoside and Nucleotide Derivatives of Pyrazofurin

monophosphate by the cellular adenosine kinase.14 5'Monophosphates of both pyrazofurin and ribavirin inhibit aminoimidazolecarboxamide ribonucleotide (AICAR) tran~formy1ase.l~ Pyrazofurin has been found to inhibit the growth of a number of different experimental tumor cell lines both in vitro and in vivo.16 Pyrazofurin completely inhibited the growth of Walker carcinosarcoma 256." Mammary carcinoma 755, Gardner lymphosarcoma, and X5563 plasma cell myeloma were inhibited more than 50% by pyrazofurin." Pyrazofurin has been studied clinically as an anticancer agent in man.4J2J6Jg Pyrazofurin was well tolerated by most patients at doses of 100 mg/m2 following iv administration. Infusion of pyrazofurin to leukemic patients resulted in severe, but reversible, toxicity. In several patients, mucositis, leucopenia, and anemia were observed,ls which limit pyrazofurin's general usage as an antitumor agent. The second active factor produced by the strain of Streptomyces candidus was subsequently isolated and characterized as the a-anomer of pyrazofurin (pyrazofurin B).20 Since the original report' of the isolation of pyrazofurin in 1969, the chemical synthesis of both a- and 0-anomers of pyrazofurin, by widely different routes, has been documented.21s26 The syntheses of 2'-deoxypyrazofurin,21the carbocyclic analogue of pyra~ofurin,~' as well as the Nl-benzyP and 1,4-dimethy120derivatives of pyrazofurin have been reported. We initiated a study of the structure and biological activity relationship of pyrazofurin and related nucleosides and nucleotides with the hope of finding a derivative with less toxicity. Now we report the chemical synthesis of several selected nucleoside and nucleotide derivatives of pyrazofurin. Pyrazofurin should be metabolized to its 5'-monophosphate by a cellular kinase to exhibit the potent antiviraljantitumor activity.'lJ2J4 Although pyrazofurin 5'-monophosphate (17) was synthesized en~ymatically~?~ by the enzyme nucleoside phosphotransferase in the presence of p-nitrophenyl phosphate as a phosphate donor, the chemical synthesis of 17 has not been previously r e p ~ r t e d . ~ Chemistry. Because of the electron-densepyrazole ring system, the chemistry of pyrazofurin is quite different from that of ribavirin (3) or even bredinin (2). The apparent pK, (in water) of pyrazofurin is 6.7. It was visualized that ~

(13) Zimmerman, T. P.; Deeprose, R. D. Biochem. Pharmacol. 1978, 27, 709. (14) Jakubowski, A.; Lehman, C.; Moyer, J.; Handschumacher, R. E. Proc. Am. Assoc. Cancer Res. 1977,18, 217. (15) Worzalla, J. F.; Sweeney. M. J. Proc. Am. Assoc. Cancer Res. 1978, 19, 56. (16) Williams, R. H.; Hoehn, M. U.S. Patent 3802999, 1974. (17) Sweeney, M. J.: Davis, F. A.: Gutowski, G. E.: Hamill. R. L.:' Hoffman, D. H.; Poore, G. A. Cancer Res. 1973,33,2619. (18) Ohnuma, T.; Holland, J. F. Cancer Treat. Rep. 1977,61, 389. (19) Cummings, F. J.; Steller, R. G.; Kaplan, H. G.; Calabresi, P. Cancer Treat. Rep. 1977, 63, 1363. (20) Gutowski, G. E.; Chaney, M. 0.;Jones, N. D.; Hamill, R. L.; Davis, F. A.; Miller, R. D. Biochem. Biophys. Res. Commun. 1973, 51, 312. (21) Farkas, J.; Flegelova, Z.; Sorm, F. Tetrahedron Lett. 1972, 2279. (22) DeBernardo, S.; Weigele, M. J. Org. Chem. 1976, 41, 287. (23) Buchanan, J. G.; Stobie, A.; Wightman, R. H. J . Chem. SOC., Perkin Trans. 1 1981, 2374. (24) Katagiri, N.; Takashima, K.; Kato, T. J . Chem. SOC.,Chem. Commun. 1982, 664. (25) Katagiri, N.;Takashima, K.; Kato, T. Nucleic Acids Res. 1982, 115, 37. (26) Katagiri, N.; Takashima, K.; Haneda, T.; Kato, T. J . Chem. SOC.,Perkin Trans. 1 1984, 553. (27) Just, G.; Kim, S. Tetrahedron Lett. 1976, 1063.

Scheme I

-

no H2NtlIJJ

H

H

1 pyrazofurln

269

;$no

n

4

1 \

the synthesis of the desired nucleoside and nucleotide derivatives of pyrazofurin might be realized by protecting the acidic hydroxyl group of the aglycon moiety. Benzylation of free pyrazofurin (1) with benzyl bromide in aqueous acetonitrile in the presence of Na2C0, gave 4-0benzylpyrazofurin (4) in excellent yield, which was found to be a useful intermediate for subsequent reactions (Scheme I). Compound 4 is indeed the 4-0-benzyl and not the N-benzyl derivative of pyrazofurin as established by comparison of the UV absorption spectrum [A, (pH 1) 245 (sh) nm; ,A, (pH 11) 243 nm] of 4 with that reported for l-benzylpyrazofurin21[A, (pH 1)232 and 268 nm; A,, (pH 11)239 and 312 nm]. Methylation of 4 with diazomethane and subsequent removal of the benzyl group by catalytic hydrogenation of the intermediate 5 with Pd/C in a hydrogen atmosphere gave 1-methylpyrazofurin (l-methyl-4-hydroxy-3-~-~-ribofuranosylpyrazole-5carboxamide, 8). The structural assignment of 8 was confirmed by single-crystal X-ray analysis. Direct methylation of unprotected 1with diazomethane in dioxane gave the dimethylated derivative l-methyl-4methoxy-3-~-~-ribofuranosylpyrazole-5-carboxamide (7) in 92% yield (Scheme I). Although compound 7 is claimed to have been synthesized, neither experimental details nor physicochemical data has been reported.20 Since N2methylation cannot be ruled out, the structure of 7 was established by single-crystal X-ray analysis. Methylation of 1 with methyl iodide in dimethylformamide in the presence of K2C03 readily gave 4-methoxy-3-P-~-ribofuranosylpyrazole-5-carboxamide(6) as the major product (>95%), along with a trace amount of 7. The mixture of 6 and 7 was separated on an open-bed silica gel column. In an effort to develop a synthetic procedure that would lead to pyrazofurin derivatives modified at the 5-position, the synthesis of 4-hydroxy-3-@-~-ribofuranosylpyrazole-5carbonitrile (10) was investigated (Scheme 11). Simple acetylation of 1 with acetic anhydride in the presence of 4- (dimethy1amino)pyridine (DMAP) at ambient temperature gave the pentaacetyl derivative 4-acetoxy-3-(2,3,5tri- O-acetyl-p-~-ribofuranosyl) -l-acetylpyrazole-5-carboxamide (9). Compound 9 has been listed in the patent l i t e r a t ~ r e , but ~~,~ no~ physicochemical data is given. Dehydration of 9 with phosgene in a mixture of pyridine and dichloromethane at room temperature gave 4-acetoxy-3- (2,3,5-tri-0-acetyl-/3-~-ribofuranosyl)-l-acetyl(28) Gutowski, G. E. U S . Patent 3 960836, 1976. (29) Gutowski, G. E. US.Patent 4053 689, 1977.

270 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2

Scheme I1

Petrie et al.

Scheme 111

--

--

NC+” H&’

Ac

NOAc

AcO

HO

”

En0 II NaO-P-0

bH

-

HO OH 4

io

17

18

OEt HO OH

HO OH

i9a,R =En

2 0 a , R = Bn

b,R:H

b,RrH

Z l a , R = Bn b,R= H

convert amides to thioamides, including sensitive aminoand hydroxy-containing corn pound^.^^ Treatment of the tetraacetyl derivative of 4 with Lawesson’s reagent in boiling dioxane gave the unstable thioamide 14, which without isolation was deacetylated with MeOH/NaOMe to yield a mixture of 4-(benzyloxy)-3-/3-D-ribofuranosylpyrazole-5-thiocarboxamide (15) and the corresponding nitrile derivative (16). Reductive cleavage of the benzyl pyrazole-5-carbonitrile, which on subsequent deacetylation ether 15 with either sodium in liquid ammonia or sodium with methanolic sodium methoxide gave the desired 10 as na~hthalene,~ in THF once again gave an intractable rea crystalline solid. Treatment of 10 with free hydroxylaction mixture from which the desired debenzylated 15 amine in boiling dioxane afforded a mixture of compounds could not be isolated. When the pentaacetyl derivative from which the isolation of the deprotected carboxamid9 was treated with Lawesson’s reagent and subsequently oxime derivative of 1 was rather difficult. In an attempt deblocked with either MeOH/NH, or MeOH/NaOMe, a to isolate the carboxamidoxime derivative, the mixture was mixture of 10 and the presumed thioamide are formed. On reacetylated with acetic anhydride in the presence of standing the solid residue converts exclusively to 10 with DMAP to obtain 4-acetoxy-3-(2,3,5-tri-O-acetyl-P-D-ribo- the elimination of H,S. furanosy1)-1-acetylpyrazole-5-N-acetoxycarboxamidoxime Compound 4 served as a versatile starting material for phosphorylation studies (Scheme 111). Thus phosphory(11). However, our attempts to deacetylate 11 with MeOH/NH, or MeOH/NaOMe under a variety of exlation of unprotected 4 with phosphoryl chloride in triperimental conditions were unsuccessful and resulted in methyl phosphate, according to the general procedure of the formation of unidentifiable products. Yoshikawa et al.,34 provided 4-(benzyloxy)-3-/3-~-riboA similar dehydration of 1,2’,3’,5’-tetraacetyl derivative furanosylpyrazole-5-carboxamide5’-phosphate (17), isoof 4, which in turn was obtained by the acetylation of 4 lated as the disodium salt. The purity and structure of with Ac,O/DMAP, gave the corresponding carbonitrile 17 was confirmed by elemental and ‘H NMR analysis. derivative. Further treatment of the intermediate carboCatalytic (Pd/C) debenzylation of 17 in an atmosphere of nitrile with MeOH/NaOMe and subsequent reaction with hydrogen readily gave the desired 4-hydroxy-3-P-~-ribohydroxylamine furnished 4-(benzyloxy)-3-P-~-ribofuranosylpyrazole-5-carboxamide5’-monophosphate (pyrazofurin 5’-phosphate, 18), which represents the first refuranosylpyrazole-5-carboxamidoxime(12), which was isolated and fully characterized. Catalytic hydrogenation ported chemical synthesis of 18. of 12 readily provided 4-hydroxy-3-P-~-ribofuranosyl- Although pyrazofurin should be converted to its 5’monophosphate (18) by a cellular kinase to exhibit biopyrazole-5-carboxamidine (13). It is of particular interest logical activity, the direct administration of preformed 18 that the corresponding carboxamidine derivative of ribavirin (l-~-~-ribofuranosyl-1,2,4-triazole-3-carboxamidine)~ may not be practical because of the ionic nature of the phosphate moiety which is responsible for poor cellular is a competitive, reversible inhibitor of inosine phosphorolysis by human lymphoblast purine nucleoside phosp e n e t r a t i ~ n .A ~ ~practical approach may be to utilize the Y-(O-alkyl phosphate) and cyclic phosphate derivatives phorylase but is not a substrate for this en~yrne.~’Since of pyrazofurin in the hope that these lipophilic prodrug pyrazofurin bears structural similarity to ribavirin and has forms would enter cells by passive diffusion and thereupon a C-C glycosyl linkage that is stable to enzymatic hybe cleaved enzymatically to the free nucleotide. Consedrolysis, the amidine derivative 13 might be expected to quently the chemical synthesis of such nucleotide derivabe an even more potent inhibitor of the phosphorylase tives of pyrazofurin was undertaken. enzyme. Lawesson’s reagent [2,4-bis(4-methoxyphenyl)1,3-dithia-2,4-diphosphetane 2,4-disulfide] introduced in 1978, (32) Scheibye, S.; Pedersen, B. S.; Lawesson, S.-0. Bull. SOC.Chim. has been shown to be a mild and effective reagent to (30) Witkowski, J. T.; Robins, R. K.; Khare, G. P.; Sidwell, R. W. J. Med. Chem. 1973, 16, 935. (31) Willis. R. C.: Robins. R. K.: Seeemiller. J. E. Mol. Pharmacol. 1

1980, IS, 287.

-

Belg. 1978, 87, 229, 299. (33) Philips, K. D.; Horwitz, J. P. J . Org. Chem. 1975, 40, 1856. (34) Yoshikawa, M.; Kato, T.; Takenishi, T. Tetrahedron Lett. 1967, 5065. (35) Plunkett, W.; Lapie, L.; Ortiz, P. J.; Cohen, S. S. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 73.

Journal of Medicinal Chemistry, 1986, Vol. 29, N o . 2 271


Table I. Crystal Data and Experiment Parameters compd

7

8

1

20a

formula

C9H13N306*H20

C10H16N306

C11H17N306

F(000)

584

576

304

C18H24N309P”20 484

space group

~12121

El2121

El

El

A b, A c, A

6.734 (3) 7.000 (2) 25.283 (13) 90 4 1191.8 1.55 0.65 0.89 0.051 0.027 0.27 1513 1091 368

a,

0, deg Z

v, A3

ddcd

sin e / A cm-’ R p,

Rw

max peak in final difference map, e A-3 total data unique obsd data unobsd data

Scheme IV

no-

& 1 3+“d+y ‘ N’

n-Lu

HO OH 17

LH

22

7.334 (2) 7.676 (3) 21.111 (10) 90 4 1183.8 1.54 0.65 0.83 0.043 0.029 0.12 1572 1304 217

7.664 (4) 7.479 (3) 11.404 (5) 100.33 (4) 2

651.7 1.47 0.70 0.77 0.051 0.044 0.36 2217 1878 135

8.403 (2) 6.916 (2) 20.311 (5) 100.97 (2) 2 1158.8 1.29 0.70 1.28 0.062 0.023 0.47 3750 2548 838

pyrazole-5-carboxamide 3’,5’-(cyclic)phosphate (%la), which on subsequent catalytic hydrogenation gave pyrazofurin 3’,5’-(cyc1ic)phosphate (21b) (Scheme 111). The appearance of a singlet for the anomeric proton in the ‘H NMR spectrum of 21b at 6 4.89 indicated the formation of the cyclic phosphate ring.39 Analytical HPLC also indicated compound 21b to be free of 18. It has recently been observed that the synthetic oncolytic carbon-linked nucleosides tiazofurin (2-p-~-ribofuranosylthiazole-4-carboxamide)40and selenazofurin (2~-~-ribofuranosylselenazole-4-carboxamide),~~ both synthesized and reported from our laboratories, are phosphorylated enzymatically to the corresponding 5’-phosp h a t e ~ which , ~ ~ in the presence of ATP and Mg2+ are further converted to the NAD analogues.43*44These NAD analogues are powerful inhibitors of IMP deh y d r ~ g e n a s e However, . ~ ~ ~ ~ ~ the NAD pyrophosphorylase in P388 lymphoblasts did not convert ribavirin to the corresponding NAD which is probably why ribavirin has relatively weak antitumor activity compared to that of tiazofurin and selenazofurin. In view of these observations, the synthesis of the NAD analogue of pyrazofurin (PAD) was of particular interest. Treatment of 17 (free acid) with the activated form of AMP, adenosine 5’-phosphoric di-n-butylphosphinothioic anhydride (22) in anhydrous pyridine in the presence of AgNO,, according to the general procedure of Furusawa and co-workers,46gave over 90% yield of the blocked PAD analogue 23a. Compound 23a was purified on a DEAESephadex column using a gradient of aqueous NH4HC03. Debenzylation of the benzyloxy function of 23a with hy-

e*1!io j;oy$ tN& H

n

NAN

H

no

NHr 230,R

n

= Bn

b,R=H

Phosphorylation of 4 with phosphoryl chloride in trimethyl phosphate a t low temperature, followed by quenching the reaction mixture with cold anhydrous ethano1,36 gave a product identified as 4-(benzyloxy)-3-&~ribofuranosylpyrazole-5-carboxamide5’-O-(ethyl phosphate) (19a). Debenzylation of 19a with Pd/C (20%) in the presence of formic acid gave 5’-0-(ethyl phosphate) of pyrszofurin (19b). The structure of 19b was assigned on the basis of its ‘H NMR (in Me2SO-d6,multiplet centered at 6 3.92 with POCH coupling of 7.42 Hz)~’and by elemental analysis. However, a similar phosphorylation of 4 with POCl,, followed by quenching the reaction mixture with methanol, gave 4-(benzyloxy)-3-@-~-ribofuranosylpyrazole-5-carboxamide5’-(di-O-methyl phosphate) (20a). Hydrogenation of 20a with Pd/C in the presence of formic acid afforded a good yield of pyrazofurin 5’-(di-O-methyl phosphate) (20b). The formation of the 5’4di-O-methyl phosphate) derivative 20a under these conditions was surprising. Therefore it was deemed desirable to unequivocally assign the structure of 20a by single-crystal X-ray analysis. Finally the dicyclohexylcarbodiimide (DCC) mediated cyclization of the nucleotide 17, according to the general procedure of Khorana and c o - w o r k e r ~ ,gave ~ ~ 4-(benzyloxy)-3-/3-~-ribofuranosyl(36) Kim, S-H.: Rosowskv. _ .A. J. Carbohvdr. Nucleosides Nucleotides 1979; 6, 229. (37) Ramirez, F.; Madan, 0. P.; Desai, N. B.; Meyerson, S.; Banas, E. M. J. Am. Chem. SOC.1963,85, 2681. (38) Smith, M.; Drummond, G. I.; Khorana, H.G. J. Am. Chem. SOC.1961, 83, 698.

(39) Revankar, G. R., Robins, R. K. “Handbook of Experimental Pharmacology; Nathanson, J. A., Kebabian, J. W., Ed.; Springer-Verlag: Berlin, 1982; Vol. 5811 p 17. (40) Srivastava, P. C.; Pickering, M. V.; Allen, L. B.; Streeter, D. G.; Campbell, M. T.; Witkowski, J. T.; Sidwell, R. W.; Robins, R. K. J. Med. Chem. 1977,20, 256. (41) Srivastava, P. C.; Robins, R. K. J. Med. Chem. 1983,26,445. (42) Kuttan, R.; Robins, R. K.; Saunders, P. P. Biochem. Biophys. Res. Commun. 1982, 107, 862. (43) Cooney, D. A.; Jayaram, H.N.; Gebeyehu, G.; Betts, C. R.; Kelley, J. A.; Marquez, V. E.; Johns, D. G. Biochem. Pharmacol. 1982,31, 2133. (44) Jayaram, H. N.; Ahluwalia, G. S.; Dion, R. L.; Gebeyehu, G.; Marquez, V. E.; Kelley, J. A,; Robins, R. K.; Cooney, D. A.; Johns, D. G. Biochem. Pharmacal. 1983, 32, 2633. (45) Gebeyehu, G.; Marquez, V. E.; Cott, A. V.; Cooney, D. A.; Kelley, J. A.; Jayaram, H. N.; Ahluwalia, G . S.; Dion, R. L.; Wilson, Y. A.; Johns, D. G. J.Med. Chem. 1985,28,99. (46) Furusawa, K.; Sekine, M.; Hata, T. J. Chem. SOC.,Perkin Trans. 1 1976, 1711.


Petrie et al.

A

N1

NZ

Cd

Figure 2. Compound 8.


drogenation in the presence of Pd/C gave PAD (23b), which was again purified on a DEAE-Sephadex column using the NH,HC03 gradient. Complete removal of NH4HCO3salt was achieved by repeated lyophilization and the free acid form of PAD was obtained by cation-exchange chromatography. All the chemical and spectral data are consistent with the structure of PAD as assigned. Single-Crystal X-ray Diffraction Analyses of Pyrazofurin ( l ) , 7,8, and 20a. Suitable crystals of 1, 7,8, and 20a were chosen for single-crystal X-ray diffraction ~tudies.4~ Data for the determination of lattice parameters and the structural study for each compound were obtained with a Nicolet P3 autodiffractometer utilizin graphite monochromated Mo Kcr radiation (A = 0.71069 ). Lattice parameters for each crystal were obtained by using a least-squares technique of 15 centered 28 values. Singlecrystal data for each crystal was measured by using a variable-speed 8-28 scan technique. Three standard reflections were measured every 97 reflections to check for crystal and electronic stability. In all four studies there was no indication of instability. All reflections with I < 2a(4 were considered to be unobserved. Experimental and crystal data are summarized in Table I. The four structures were solved by using direct methods and refined by least-squares techniques. The difference maps of 1 and 20a showed the presence of water of hydration in each of the crystal structures. Non-hydrogen atoms were refined anisotropically while hydrogen atoms, which were located in difference maps were refined isotropically. Exceptions to the refinement process occurred

R

(47) The X-ray structure of 1 was reported at a meeting (Jones, N. D.; Chaney, M. 0.9th International Congress of Crystallography, Kyoto, Japan, (1972, Abstracts, p S-48) but to our

knowledge has not been published. For that reason and because of its importance to this study, the X-ray structure of l was determined and is reported here.


in 20a where the phenyl group and the methyl groups were refined as rigid bodies and in 7 where the methyl group bonded to N1 was also refined as a rigid body. Also in 20a, O5’PO2 was badly disordered. The disorder was resolved by using a difference map and during further refinement the positional parameters of the partial atoms were held constant, the occupancy factors were fixed at 0.60 (05’P02a)and 0.40 (O5’PO2b),and the partial atoms were refined anisotropically. The R values for the four crystals are listed in Table I. Weights based on counting statistics


Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2 273

Table 11. Intramolecular Hydrogen Bond Data compd D-He-A D-A, 8, H-A, 8,

D-H-A, dep N6-H1N6.*.04 2.895 (5) 2.28 (4) 129 (3) 7 N6-H1N6**-04 2.754 (3) 2.122 (2)” 132.6 (2)’ 131 (3) 8 N6-H1N6-*04 2.786 (3) 2.00 (3) 20a N6-H1N6.-04 2.938 (4) 2.31 (3) 124 (3) 1 04-H04*-05’ 166 (4) 2.642 (4) 1.92 (3) 8 04-H04-*05’ 149 (3) 2.674 (3) 1.93 (3) “The low esd values reported result from the fact that the positional parameters of H’N6 were held fixed in the refinement. More reasonable uncertainties for these values would be *0.004 8, for the H-A distance and *4’ for the D-H-A angle. 1

Figure 4. Compound 20a.

were used in the refinement of the four compounds. All computer calculations performed in this study were made by using SHELX-76.48 Computer drawing showing structural formulas, conformation, atom labels, and interatomic bond distances are shown in Figures 1-4. The estimated standard deviations on all bond lengths are between 0.004 and 0.006 A. The hydrogens of the carbohydrate moiety are omitted for clarity. In 20a, 05’PO is represented by the atom in the more populated site. All drawings were made by using the SHELXTL4’ plotting program, which is part of the Nicolet R3 system. All four compounds exist in the /3-anomeric configuration. There are extensive intermolecular hydrogen bonding networks in all four structures. These generally involve all of the alcoholic hydrogens of the carbohydrate, plus in some cases one of the hydrogen atoms bonded to the carboxamide nitrogens and the hydrogen atoms of waters of hydration when present. In addition there are interesting intramolecular hydrogen bonds in all four compounds. A hydrogen bond common to all four molecules is the N6-HN1-04 interaction. The intramolecular hydrogen bond data is summarized in Table 11. Another interaction which occurs in 1 and 8 is the 04-H04-05’ hydrogen bond. A similar hydrogen bond is not possible in 7 and 20a as the H04 has been replaced by a substituent in each structure. The data in Table I1 clearly shows that the 04-05’ hydrogen bonds are significantly shorter and therefore stronger than the N6-H1N6--04 interactions. This is likely due to the rather fixed positional relationship of N6 and 0 4 established by the aglycon. The 04-05’ (48) Sheldrick, G. M. SHELX-76, “A Program for X-Ray Crystal Structure Determination“,University of Cambridge, England, 1976. (49) Sheldrick, G. M. SHELXTL, 4th revision, “An Integrated System for Solving, Refining and Displaying Crystal structures from Diffraction Data”, University of Gottingen, Federal Republic of Germany, 1983.

spatial relationship is much more flexible due to the presence of the carbohydrate moiety. It is interesting to note that the 04-02’ hydrogen bond is reported20 in the a-conformer of 1. The 0-0interatomic distance of 2.551 (4) A is shorter than those found in 1 and 8, indicating a strong hydrogen bond. The a-conformation brings 02’ rather than 05’ close to 0 4 . The same authors report a 04-05’ distance of 2.61 A in 1 in a footnote.20This value, although slightly shorter than the value reported in this study, supports the presence of a rather strong hydrogen bond between 0 4 and 05’. Biological Evaluations Antiviral Activity. The nucleoside and nucleotide derivatives of pyrazofurin synthesized during this study were tested against parainfluenza type 3 (ParaS), measles, vaccinia (w),and herpes simplex type 2 (HSV2) viruses in cell culture in parallel with pyrazofurin (1) and ribavirin (3). The results of the antiviral evaluation are shown in Table 111. It is evident that some of the nucleotide derivatives of l retain significant antiviral activity in vitro. Pyrazofurin (1) exhibited pronounced antiviral activity against all the viruses tested with ED50 values 10-50 times lower than that of ribavirin (3). The 5’-monophosphate (18) and the NAD analogue (23b) of pyrazofurin were active at concentrations very similar to that of 1. However, the 5’-(O-ethyl phosphate) (19b) had lower antiviral activity with EDm concentrations 5-25 times higher than that of 18. The Y-(di-O-methylphosphate) (20b) was even less active with ED50 values approximately 10 times higher than that of 19b and 50-250 times higher than that of 18. The 3’,5’-(cyclic)phosphate(21b) was also considerably less active than 18 with ED,, values approximately 10-100 times greater than that of 18 or 1. Pyrazofurin derivatives with modifications at either the 1-, 4-, or 5-positions (4, 6-8, 10, 12, 13, 15, and 17) were inactive or exhibited drastically reduced antiviral activity as compared to that of 1. The maximum tolerated concentration (MTC) of pyrazofurin was 1.6 X M for the confluent monolayers of Vero cells used for the antiviral evaluations. Under identical conditions, the 5’-monophosphate (18) did not exhibit toxicity at concentrations as high as 5.0 X M and may therefore have some therapeutic advantage over 1. The NAD analogue 23b was also considerably less toxic than 1, with an MTC of 5.0 X M. These observations indicate that compounds 18 and 23b may have better antiviral potential than pyrazofurin. Cytostatic Activity. All the newly synthesized nucleoside and nucleotide derivatives of pyrazofurin were also tested for their inhibitory effects on the growth of L1210 and P388 leukemias, B16 melanoma, and Lewis Lung carcinoma in vitro. The results of the evaluation of these cytostatic activity are summarized in Table IV. Pyrazofurin, its 5’-monophosphate (18), and the NAD analogue (23b), all exhibited significant inhibitory effects on the growth of L1210 and P388 leukemias and Lewis Lung


Petrie et al.

Table 111. Comparative in Vitro Antiviral Activity of Pyrazofurin (11, Ribavirin (31, and Certain Nucleoside and Nucleotide Derivatives of Pyrazofurin in Vero Cells E L bM MTC,” M Para3 measles VV h5v2 >5.0 x 10-3 >5.0 x 10-3 1.3 x 10-3 >5.0 x 10-3 >5.0 x 10-3 6 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 2.8 x 10-3 >5.0 x 10-3 7 5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 5.0 x 10-3 3.2 x 10-3 8 x . 0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 1.6 x 10-3 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 5.0 x 10-3 5.0 x 10-3 >5.0 x 10-3 10 12 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 1.6 x 10-3 5.0 x 10-3 >5.0 x 10-3 3.1 x 10-3 13 >5.0 x 10-3 >5.0 x 10-3 5.0 x 10-4 2.1 x 10-3 2.8 x 10-3 15 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 >5.0 x 10-3 17 >5.0 x 10-3 6.3 X 10” 2.0 x 10-5 4.0 X 10” 2.3 x 10-5 18 >5.0 x 10-3 8.0 x 10-5 9.7 x 10-5 2.6 x 10-5 5.9 x 10-4 19b 20b >5.0 x 10-3 1.0x 10-3 1.4 x 10-3 2.8 x 10-4 >5.0 x 10-3 5.0 x 10-4 25.0 x 10-3 2.5 x 10-4 1.3 x 10-4 21b >5.0 x 10-3 5.0 x 10-3 5.0 x 10” 1.6 x 10-5 9.0 x 10“ 1.0x 10-5 23b 1 1.6 x 10-3 3.0 X lo4 9.6 X 10” 2.8 X lo4 2.0 x 10-5 >5.0 x 10-3 1.6 x 10-4 9.9 x 10-5 9.0 x 10-5 3 1.9 x 10-4 a The maximum tolerated concentration (MTC) is the lowest concentration of compound that produced microscopically visible cytotoxicity assessed as described in the Experimental Section. bThe ED, is the concentration of compound that produced 50% inhibition of viral CPE as compared with nondrug controls. Values represent averages of two to three separate determinations. compd 4

Table IV. In Vitro Cytostatic Activity of Pyrazofurin (1) and Certain Nucleoside and Nucleotide Derivatives of Pyrazofurin ID,,” M compd L1210 P388 Lewis Lung B16 melanoma 4 7.5 x 10-5 6.2 x 10-5 b b 6 2.9 x 10-5 4.0 x 10-5 b b 7 > L O x 10-4 >1.0 x 10-4 b b 8 1.5 x 10-5 1.7 x 10-5 b b 10 1.3 x 10-5 4.5 x 10-5 2.8 x 10-5 7.2 x 10-5 12 >LO x 10-4 >LO x 10-4 b b > L O x 10-4 8.9 x 10-5 >LO x 10-4 13 7.9 x 10-5 15 > L O x 10-4 >1.0 x 10-4 >LO x 10-4 >LO x 10-4 17 1.3 x 10-4 9.4 x 10-5 b b 18 2.7 x 10-9 1.0 x 10-9 5.0 X lo* 2.4 x 10-7 19b 3.9 x 10-9 3.2 x 10-9 7.0 X 2.3 x 10-7 20b 5.8 x 10-9 1.2 x 10-8 1.0 x 10-8 2.3 X lo4 21b 2.5 X 10” 4.4 x 10” 1.7 X 10“ 6.3 X lo4 23b 4.0 x 10-9 8.8 X 4.8 x 10-9 6.0 x 10-7 1 3.2 x 10-9 3.3 x 10-9 1.8 x 10-9 6.3 X “Inhibitory dose 50 (IDw) is the concentration of the compound in the culture media that produced 50% of the tumor cell growth as compared to the untreated controls. Values represent averages of two to three separate determinations. Not determined.

carcinoma cells in vitro, with similar ID,, values in the nanomolar concentration range. However, B16 melanoma cells were less sensitive to growth inhibition by these compounds. The ID,, value of pyrazofurin was approximately 20-35 times higher against B16 melanoma than against other cell lines. Compounds 18 and 23b were less effective than 1 in inhibiting B16 melanoma with ID,, values 4-10 times higher than that of pyrazofurin and 50-250 times higher than their ID,, values against the other cell lines tested. The 5/-(O-ethylphosphate) (19b)had approximately the same cytostatic activity as the 5’-monophosphate (18), while the 5’-(di-O-methyl phosphate) (20b) had an ID,, value 2-12 times higher than that of 18. Modification at the 1-,4-, or 5-position of 1 (compounds 4,6,8, 10, 13, and 17) considerably reduced the cytostatic activity in cell culture, and compounds 7, 12, and 15 were inactive against L1210 and P388 leukemias. Experimental Section Melting points were taken on a Thomas-Hoover capillary melting point apparatus and are uncorrected. Nuclear magnetic resonance (lH NMR) spectra were determined at 89.6 MHz with a JEOL FX 9OQ spectrometer. The chemical shift values are expressed in 6 values (parts per million) relative to tetramethylsilane as an internal standard. The presence of water as indicated by elemental analysis was verified by ‘H NMR. Infrared spectra (IR) were obtained on a Beckman Acculab 2 spectro-

photometer and ultraviolet spectra (UV; sh = shoulder) were recorded on a Cary Model 15 spectrophotometer. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN, and Robertson Labs, Florham Park, NJ. Thinlayer chromatography (TLC) was run on silica gel 60 F-254 plates (EM Reagents). E. Merck silica gel (230-400 mesh) was used for h h column chromatography. preparative liquid chromatography (LC) was run utilizing the Waters Prep 500 LC system. All solvents used were reagent grade and were dried over molecular sieve (4A). Detection of components on TLC was by UV light and with 10% H2S04 in MeOH spray followed by heating. Evaporations were carried out under reduced pressure with the bath temperature below 30 “C. 4-(Benzyloxy)-3-~-~ribafuranosylpyrazole-5-car~xamide (4). To a solution of pyrazofurin (1; 10.36 g, 40 mmol) in water (200 mL) containing Na2C03(4.24 g, 40 mmol) was added benzyl bromide (7.6 g, 60 mmol) in CH&N (200 mL), all a t once with stirring. After stirring for 18 h at room temperature, the solution was evaporated to dryness. The residue was dissolved in a minimum amount of hot water and allowed to stand a t room temperature overnight. The white crystals that deposited were collected by filtration to yield 8.4 g. The mother liquor was chromatographed on a reverse-phase silica gel column (Prep 500) using 5% MeOH in water as the eluent. The fractions containing the desired product were pooled and evaporated to dryness. The residue was triturated with ether (50 mL) and filtered to give 4.1 g of white crystals. Recrystallization of the combined solids from aqueous acetonitrile afforded 12.2 g (87.4%) of pure 4; mp 163-165 OC; IR (KBr) Y 1050 (=COC), 1650 (C===O),2900-3430 (OH, NH2) (pH 1) 245 (sh) nm (c 3200); UV A,, (pH 7 ) 245 cm-’; UV A,



bands were eluted, which were evaporated to dryness. The first band gave the title compound as amorphous foam, 9.8 g (52.2%); IR (KBr) u 1685 (C=O of amide), 1785 (C=O of acetyl) cm-'; (MeOH) 252 nm (E 29100); 'H NMR (CDCl,) 6 2.08-2.12 UV ,A, l-Methyl-4-hydroxy-3-~-~-ribofuranosylpyrazole-5-(3 s, 9, 3 OAc), 2.36 (s, 3, C4-acetoxy), 2.70 (s, 3, NAc), 5.64 (d, 1, J = 3.10 Hz, C1,H), 6.20, 6.79 (2 br s, 2, CONH,), and other carboxamide (N'-Methylpyrazofurin, 8). A solution of 4 (2.0 sugar protons. Anal. (C19H23N3011-H20) C, H, N. The second g, 7.7 mmol) in dioxane (100 mL) was saturated with diazomethane product was found to be tetraacetylpyrazofurin. and the mixture was allowed to stand a t room temperature for 4-Hydroxy-3-~-~-ribofuranosylpyrazole-5-carbonitrile 18 h. Acetic acid was added to destroy excess diazomethane and (10). To a solution of 9 (9.4 g, 2 mmol) (or a mixture of 9 and the solution was evaporated to dryness. The residue containing 1-methyl4(benzy~oxy)-3-P-~ribofuranosylpyrazole-5-~b~detetraacetylpyrazofurin) in CH,Cl, (100 mL) containing pyridine (5 mL) was added phosgene in toluene (12.5%, 50 mL) dropwise (5)was dissolved in MeOH (75 mL) and hydrogenated on a Parr at a rate that maintained a constant temperature. The resulting hydrogenator a t 45 psi for 5 h in the presence of Pd/C (lo%, 100 dark red solution was poured onto crushed ice (100 g) and exmg). The mixture was filtered through a Celite pad and washed tracted with water (3 X 50 mL). The combined water extracts with hot MeOH (3 X 25 mL), and the combined filtrates were were then extracted once with CH2Cl2(100 mL). The organic evaporated to a syrup. The syrup was dissolved in CH3CN (10 mL) and allowed to stand a t room temperature for several days. phases were combined and dried over anhydrous Na2S04. The The title compound crystallized out, 0.30 g. Additional amount solution was filtered and evaporated to dryness. The residue was subjected to silica gel flash chromatography (4.1 X 15 cm column, of 8 was obtained after chromatographing the mother liquor on a silica gel column (2 X 25 cm) using CH2C1,/MeOH (9:1, v/v) acetone/hexane, 1:4, v/v) to give pure 4-acetoxy-3-(2,3,5-tri-Oacetyl-~-~-ribofuranosyl)-l-acety~pyrazole-5-c~bonitr~~e as a gum as the eluent. Total yield of 8 was 0.70 g (52.5%, from aqueous (8.0 g, 88.6%). acetonitrile); mp >195 "C dec; IR (KBr) u 1650 (C=O), 2930 (pH 1) 230 nm The above gum (5.0 g, 11 mmol) was dissolved in MeOH (100 (NCH,), 3360 and 3470 (OH, NH,) cm-'; UV ,A, (E 8200), 267 (5600); UV ,A , (pH 7) 232 nm (E 6500), 308 (5200); mL) and 1N NaOMe was added to pH 9. The mixture was stirred W A- (pH 11)237 nm (E 6000), 309 (8200); 'H NMR (MezSO-d6) for 16 h a t room temperature and neutralized with Doxex-50 Ht 6 3.92 (5, 3, CH,), 4.71 (d, 1, J = 6.10 Hz, C1,H), 7.22 (br s, 3, resin and the solvent evaporated. The residual syrup was trituCONH,, C4-OH),and other sugar protons. Anal. (CloH15N306) rated with anhydrous ether (50 mL). The solid was collected by C, H, N. filtration and crystallized from aqueous ethanol to yield 2.3 g 4-Methoxy-3-~-~-ribofuranosylpyrazole-5-carboxamide (86.2%) of the title compound; mp 138-139 "C; IR (KBr) u 2230 (C=N), 2950-3430 (OH, NH) cm-'; UV, , A (4-0-Methylpyrazofurin,6).Pyrazofurin (1.0 g, 3.8 mmol) in (pH 1) 257 nm (e 5100); UV A,, (pH 7) 296 nm ( e 5500);UV ,A, (pH 11) 296 nm dimethylformamide (25 mL) containing methyl iodide (0.59 g, (E 8000);'H NMR (Me2SO-ds)6 4.73 (d, 1, J = 6.44 Hz, CIrH), 4.18 mmol) and KzCO, (0.57 g, 4.18 mmol) was stirred at room and other sugar protons. Anal. (C9HllN30?)C, H, N. temperature for 24 h. The reaction mixture was diluted with 0.5 N acetic acid (25 mL) and evaporated to dryness. The residue 4-Acetoxy-3-(2,3,5-tri-O-acetyl-B-D-ribofuranosy1)-1was purified on a silica gel column (2.5 X 25 cm) using acetylpyrazole-5-N-acetoxycarboxamidoxime( 11). To a solution of 10 (2.41 g, 10 mmol) in anhydrous dioxane was added CH2C12/MeOH(9:1, v/v) as the eluent to yield 1.0 g (95.2%) of the title compound after crystallization from water; mp >210 "C free hydroxylamine (0.5 g, 15 mmol) and the mixture was heated under reflux for 1h, with the exclusion of moisture. The reaction dec; IR (KBr) u 1015 (=COC), 1670 (C=O), 3340-3440 (OH, NH,) (pH 1 and 7) 224 nm ( e 14500);UV A,, (pH 11) cm-'; UV A,, mixture was evaporated to dryness and held at high vacuum 224 nm (c 15000);'H NMR (MezSO-d6)6 3.78 (s, 3, CH,), 4.67 overnight. The dry residue was treated with acetic anhydride (25 mL) and DMAP (100 mg). The mixture was stirred a t room (d, 1,J = 6.45 Hz, Cl,H), 7.32, 7.16 (2 br s, 2, CONH2), 12.80 (br s, 1, NH), and other sugar protons. Anal. (CloH15N306~H,0) C, temperature for 5 h and evaporated to dryness. Coevaporation H, N. with absolute ethanol (3 X 25 mL) gave dry residue, which was 1-Methyl-4-methoxy-3-~-~-ribofuranosylpyrazole-5- purified on a silica gel column (2.4 X 45 cm) using toluene/ethyl acetate (7:3, v/v) as the solvent. The appropriate homogenous carboxamide (1,4-Dimethylpyrazofurin,7). To a hot solution of pyrazofurin (1.0 g, 3.8 mmol) in dioxane (free from peroxides) fractions were pooled and evaporated to dryness and the residue was triturated with CCl, (25 mL) to give crystalline solid, 2.5 g was added an ice-cold solution of diazomethane in dioxane. The reaction was monitored by TLC (silica gel, CH2C1,/MeOH, 91, (47.5%); mp 238-240 "C; IR (KBr) u 1760 and 1780 (C=O of v/v), with additional amounts of diazomethane in dioxane being acetyl), 3370, 3480 (NH,) cm-'; UV, , A (pH 1) 230 nm (E 9200); UV A, (pH 7) 230 nm (E 8700); UV A, (pH 11)300 nm ( E 6000); added. When the reaction had gone -75%, enough diazomethane 'H NMR (MezSO-d6)6 2.00, 2.01, 2.04 (3 s, 9, 3 OAc), 2.12 (s, 3, was added to impart a dark yellow color and the reaction mixture was allowed to stand in the refrigerator overnight. TLC indicated NAc), 2.30 (5, 3, C~-OAC), 2.72 (5, 3, NOAC),5.54 (s, 1, ClCH), 6.79 (br s, 2, NH,), and other sugar protons. Anal. (C21H26N4012) C, that the reaction had gone almost to completion with only small H, N. amounts of mono- and higher methylated products. The mixture was acidified with acetic acid to destroy excess diazomethane and 4-(Benzyloxy)-3-~-~-ribofuranosylpyrazole-5-carboxthen evaporated to dryness. The residual syrup was triturated amidoxime (12). A solution of 4 (2.0 g, 5.7 mmol) in anhydrous with CH3CN (10 mL) to give crystalline 7 (0.35 9). Additional pyridine (50 mL) containing acetic anhydride (10 mL) and DMAP material was obtained by chromatographing the mother liquor (25 mg) was stirred at room temperature under anhydrous con(silica gel using CH2Cl,/MeOH, 9:1, v/v, as solvent). Recrysditions for 18 h before it was poured onto ice-water (100 mL). tallization of the combined solids from water gave 1.02 g (92%) After stirring overnight, the mixture was extracted with CH,CI, (3 x 75 mL). The combined, dried (Na2S04)organic layer was of the title compound; mp 179-180 "C; IR (KBr) u 1010 (=COC), 1640 (C=O), 2920 (NCH,), 3310-3410 (OH, NHz) cm-'; UV ,A, evaporated to yield 2.85 g (96.6%) of the tetraacetyl derivative (pH 1 and 7) 228 nm (E 6200); UV ,A, (pH 11) 228 nm (t 6000); of 4 as homogeneous gum. 'H NMR (MepSO-d6) 6 3.82 (s,3, C4-OCH3), 3.96 (9, 3, NX-CH,), To a solution of the above tetraacetyl derivative (2.5 g) in 4.65 (d, 1, J = 6.45 Hz, CltH),7.18,7.70 (2 s, 2, CONH,), and other CHPC12(75 mL) containing pyridine (3.5 mL) was added dropwise a solution of phosgene in benzene (1.2 equiv). After the addition sugar protons. Anal. (Cl1H1,N3o6) c, H, N. 4-Acetoxy-3- (2,3,5-tri- 0 -acetyl-8-D-ribofuranosy1)1was complete, the reaction mixture was allowed t o warm to room acet y lpyrazole-5-carboxamide ( 0 4,N',2',3',5'-Pentaacetyltemperature for 2 h and then poured onto crushed ice (75 9). The pyrazofurin, 9). A mixture of pyrazofurin (10.36 g, 40 mmol) aqueous solution was extracted with CHzC12(3 X 50 mL). The and 4-(dimethy1amino)pyridine(DMAP, 25 mg) in acetic anhycombined, dried (Na2S04)organic layer was evaporated to dryness dride (100 mL) was stirred at ambient temperature for 18 h. The and the residue dissolved in MeOH (50 mL). NaOMe (1N) was solution was evaporated to dryness and the residue was coevaadded until pH 10 was maintained. After standing overnight, porated several times with toluene. TLC (silica gel, CHC13/ the solution was neutralized with Dowex-50 H+ resin and filtered acetone, 8:2, v/v) indicated a mixture of two products. The and the filtrate evaporated to dryness. The residue was purified mixture was purified on a silica gel column by preparative LC on a silica gel column (2.3 X 25 cm) using CHCl,/MeOH (61, v/v) techniques with acetone/hexane (4:6, v/v) as the solvent. Two as the solvent to yield the deblocked nitrile, 1.90 g. The nitrile (sh) nm (E 4200); UV A,, (pH 11) 243 nm (E 6300); 'H NMR (MezSO-ds) 6 4.72 (d, 1,J = 6.20 Hz, CltH), 5.17 ( 8 , 2, CHJ, 7.40 (m, 7, CHzC6r, and CONHz), and other sugar protons. Anal. (Ci6Hi9N306) c, H, N.

276 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 2 was dissolved in absolute ethanol to which was added 5 equiv of free NHzOH. The solution was heated under reflux for 10 h and allowed to stand at room temperature overnight. The solution was evaporated to dryness and the residue was chromatographed on a silica gel column (2.5 X 25 cm) using CHCl,/MeOH (51,v/v) as the solvent to yield 1.50 g (85.2%) of the title compound after crystallization from aqueous MeOH; mp 125-127 "C; IR (KBr) u 1075 (=COC), 2940-3300 (OH, NH,) cm-'; UV A , (pH 1)235 (pH 7) 235 nm ( e 6400); UV ,A, (pH 11) nm ( e 7300); UV ,A, 235 nm (t 6900); 'H NMR (Me2SO-d6)6 4.68 (d, 1,J = 6.45 Hz, Cl(H), 4.98 (9, 2, CH,), 5.46 (9, 2, NHz), 7.38 (m, 5, C$5), 9.55 (s, 1, NOH), 12.61 (s, 1, NH), and other sugar protons. Anal. (Ci6H2oN406) c, H, N. 4-Hydroxy-3-b-~ribof uranosylpyrazole-5-carboxamidine (13). To a solution of 12 (50 mg, 0.13 mmol) in 95% EtOH (10 mL) was added 20% Pd/C (20 mg) and the mixture was shaken on a Parr hydrogenator for 48 h at 50 psi. The mixture was fdtered on a Celite pad and the filtrate evaporated to dryness. The residue was purified on a preparative TLC silica gel plate and developed with EtOAc/EtOH/HzO (2:1:1, v/v). The appropriate band was scraped from the plate and eluted with MeOH. The solution was evaporated to dryness. The residue was dissolved in hot EtOH (95%) and filtered. On standing fluffy white needles separated, yield 24 mg (62.7%); mp 188 OC dec; IR (KBr) Y 1670 (C=NH), (pH 1)227 (sh) nm (t 8800), 3200-3400 (OH, NH,) cm-'; UV A, 276 (4400);UV A,, (pH 7) 326 nm (t 4400);UV A, (pH 11)325 295 nm (t 3000); lH NMR nm ( t 7000); isosbestic point A, (MeaO-d,) 6 4.83 (d, 1,J = 5.10 Hz, Cl,H), 7.50 (2 br s, 4, C4-OH, C=NH, NHJ, 12.50 (br s, 1,NH), and other sugar protons. Anal. (CgH14N4Og2HzO) C, H, N. 44 Benzyloxy)-3-~-~ribofuranosylpyrazole-5-thiocarboxamide (15) a n d 4-(Benzyloxy)-3-~-~-ribofuranosylpyrazole-5-carbonitrile (16). A portion of the tetraacetyl derivative of 4 from reaction 12 (3.0 g, 5.8 mmol) was dissolved in anhydrous dioxane (50 mL) containing 2,4-bis(4-methoxy2,4-disulfide (Lawesson's phenyl)- 1,3-dithia-2,4-diphosphetane reagent, 1.20 9). The solution was heated under reflux for 2 h before it was poured into ice-water (50 mL). After stirring for 30 min, the mixture was extracted with CH2C12(3 X 50 mL). The combined organic phase was dried (Na2S04)and evaporated to dryness. The residue was purified on a silica gel column (2.4 X 40 cm) using acetone/hexane (1:4, v/v) as the solvent. Fractions containing the desired product were pooled and evaporated to dryness to yield 2.5 g of the blocked thioamide (14) as a yellow foam. The solution of 14 (2.5 g) in MeOH (65 mL) was adjusted to pH 10 with NaOMe. After stirring a t room temperature for 2 h, the mixture was neutralized with Dowex-50 H+ resin and then evaporated to dryness. The residue was triturated with CHCl, (50 mL) and the solid that separated was collected and applied to a flash silica gel column. The column was eluted with CHC13/MeOH (6:1, v/v). The first nucleoside product to elute was found to be 16, which was isolated as a syrup to yield 0.15 g (70%); IR (KBr) Y 2240 ( C s N ) cm-'. The second product to elute was identified as 15, which was crystallized from aqueous ethanol to yield 1.32 g (62.3% overall yield); mp 146-148 "C; IR (KBr) u 1080 (=COC), 1200, 1500 (pH 1) 253 nm (c (C(S)N), 3300-3360 (OH, NH2) cm-'; UV A,, 6600), 297 (9500);UV A, (pH 7) 253 nm ( t 7500), 297 (11000); UV A,, (pH 11) 265 nm ( e 8800), 305 (12100); 'H NMR (MezSO-d6) 6 4.71 (d, 1,J = 5.90 Hz, CiH), 5.07 (s, 2, CH,), 7.38 (m, 5, C6H5),8.82, 9.60 (2 s, 2, C(S)NH,), 12.96 (br s, 1,"1, and other sugar protons. Anal. (C16HlsN3O5S) c, H, N, S. 4-(Benzyloxy)-3-@-~-ribofuranosylpyrazole-5-carboxamide 5'-Monophosphate Disodium Salt (17). To an ice-cold solution of 4 (2.0 g, 5.7 mmol) in freshly distilled trimethyl phosphate (50 mL) was added POCl, (2 mL). The reaction mixture was stirred at ice-bath temperature and monitored frequently by TLC. When all the starting material had disappeared (1-2 h), the mixture was poured into ice-water (200 mL). The aqueous solution was extracted with ether (3 X 50 mL) and then adjusted to pH 2 with 2 N NaOH. The solution was applied to a column of activated charcoal (50 g, acid-washed A u - ~ )and , the column was washed thoroughly with water until the eluate was salt free. The column was eluted with a solution of EtOH/H20/NH40H( l O l O : l , v/v). The fractions containing pure (TLC, silica gel, CH3CN/0.1 N "&I, 7:3, v/v) nucleotide were pooled and evaporated t o dry-

Petrie et al. ness. The residue was dissolved in a small amount of water and passed through a column of Dowex-50 H+ resin (20-50 mesh, 25 mL). The column was washed with water, and the fractions containing the nucleotide were collected. The solution was concentrated to a small volume (-20 mL) and passed through a column of Dowex 50W-X8 (20-50 mesh, Na+ form, 25 mL). The column was washed with water, and the fraction containing the nucleotide sodium salt was evaporated to dryness. The solid residue was triturated with absolute EtOH (20 mL) before the white solid was collected by filtration and dried (P205)to provide 1.50 g (55.6%) of the title compound; mp >145 "C dec; IR (KBr) Y 1050 (=COC), 1665 (C=O), 2940-3440 (OH, NH,) cm-I; UV A, (pH 1 and 7) 240 (sh) nm ( e 4000); UV ,A, (pH 11)240 (sh) nm ( e 5400); 'H NMR (Me2SO-d6)6 5.00 (s, 2, CHz), 5.09 (d, 1, J = 7.46 HZ, CiTH), 7.07, 7.21 (2 S, 2, CONH,), 7.32 (m, 5, C&), C, H, N, and other sugar protons. Anal. (C1~H18N309Na2P.HzO)

P. 4-Hydroxy-3-B-~-ribofuranosylpyrazole5-carboxamide 5'-Monophosphate (Pyrazofurin 5'-Monophosphate, 18). Compound 17 (2.0 g, 4.2 mmol), formic acid (5 mL), and 10% Pd/C (0.10 g) in MeOH (100 mL) were shaken under hydrogen atmosphere (50 psi) for 24 h. The catalyst was removed by filtration on a Celite pad and the filtrate evaporated to dryness. The residual solid was dissolved in water (50 mL) and passed through a column of Dowex-50 H+ resin (25 mL). The eluate was lyophilized to obtain off-white powder, 1.10 g (77.2%); mp 90-95 "C; IR.(KBr) u 1675 (C==O), 3200-3380 (OH, NH,) cm-'; UV A, (pH 1)257 nm (c 4300); UV A, (pH 7) 257 nm (t 3700); UV A, (pH 11) 303 nm ( e 5400); 'H NMR (MezSO-d6)6 4.73 (d, 1,J = 6.32 Hz, C1.H),7.40 (br s, 3, C4-OHand CONH,), and other sugar protons. Anal. (C9H14N30sP~11/4H20) C, H, N, P. 4-(~nzyloxy)-3-@-D-~bofuranosylpyrazole-5-carboxam~de 5'-(O-Ethyl phosphate) (19a). POCl, (2 mL) was added slowly to an ice-cold solution of 4 (2.0 g, 5.7 mmol) in freshly distilled trimethyl phosphate (25 mL). The solution was stirred for 2 h at 0-5 "C before cold anhydrous EtOH (25 mL) was added. The mixture was kept at 0 "C for 12 h. Water (100 mL) was added and the pH of the aqueous solution was adjusted to 7 with 2 N NaOH. The solution was extracted with ether (3 X 50 mL), then concentrated to -50 mL, and chromatographed on a Prep-500 reverse-phase column using 10% MeOH in 0.2 N AcOH. The fractions captaining the desired nucleotide product were pooled and evaporated to dryness. The residual syrup was triturated with ether (3 X 50 mL),which crystallized on standing for 2 weeks, to yield 1.50 g (58%) of the title compound; mp 113-115 "C; IR (KBr) Y 1020 (COC), 1100, 1160,1210 (EtOPO), 1660 (C=O), 3150-3410 (NH,, OH) cm-'; UV A,, (pH 1 and pH 7) 246 nm ( e 6400); UV A,, (PH 11) 243 nm ( e 7800); 'H NMR (MezSOd6) 6 1.14 (t, 3, J = 7.42 Hz, POCHzCH3),3.87 (m, 2, JH,p = 7.42 Hz and JH,H = 7.42 Hz, POCHZCH,), 4.70 (d, 1, J = 6.45 Hz, C1,Hj, 5.02 (s, 2, CHzC6H5),7.08, 7.40 (br s and m, 7, CONH2,CH2C,&), and other sugar protons. Anal. (Cl8Hz4N3O9P-H2O) C, H, N, P. 4-Hydroxy-3-~-~-ribofuranosylpyrazole-5-carboxamide 5'-(O-Ethyl phosphate) (19b). In the same manner as for 18, hydrogenation of 19a (1.1g) in EtOH (100mL) containing formic acid (5 mL) in the presence of 20% Pd/C (100 mg) gave the title compound, 0.75 g (85%); mp 68-71 "C; IR (KBr) u 1030 (POEt), 1190 (POH), 1660 (C=O), 3200-3400 (OH, NHz) cm-'; UV A, (pH 1) 218 nm ( e 6600), 263 (5500); UV A,, (pH 7) 263 nm ( t 5000), 307 (sh) (1600);UV A,, (pH 11) 234 (sh) nm ( e 5300),307 (8600);'H NMR (MezSO-d6)6 1.16 (t, 3, J = 7.42 Hz, CH3),3.92 (m, 2, JH,p = 7.42 Hz, CHJ, 4.74 (d, 1, J = 6.09 Hz, CInH), 7.39 (br s, 3, C4-OH and CONHz), and other sugar protons. Anal. (CiiHi~N30J')C, H, N, P. 4-(Benzyloxy)-3-~-~-ribofuranosylpyrazole-5-carboxamide 5'-(Di-O-methyl phosphate) (20a). Compound 4 (2.0 g, 5.7 mmol) was suspended in trimethyl phosphate (25 mL) and cooled to 0 OC. P0Cl3 (2 mL) was added dropwise with stirring. After 3 h, the reaction was quenched by the addition of MeOH (50 mL) and the solution was stored at 0 "C overnight. The mixture was poured into toluene (100 mL). The layers were separated, and the lower layer was again poured into toluene (100 mL). The toluene was decanted from an oily residue, which was dissolved in EtOAc (50 mL) and adsorbed onto silica gel (-15 8). The silica gel was placed on top of a silica gel column (2.5 X 40 cm) and eluted with CHCl,/MeOH (6:1, v/v). The fractions containing



NH4HC03and applied to a DEAE-Sephadex column (2.5 X 40 the desired product were pooled and evaporated to dryness. The cm) equilibrated with 0.5 M N&HCO3. The column was washed residue was crystallized from water to yield 1.70 g (62.9%) of the with 0.05 M NH4HC03(1L) and then eluted with a gradient of title compound; mp 100-102 "C; IR (KBr) v 1035 (COC), 1185 0.05 M NH4HC03(1L) to 6 N NH4HC03(1L) at a flow rate of (P), 1665 (C=O), 3290-3450 (OH, NH2) cm-'; UV ,A, (pH 1) 3 mL/min. Fractions containing the desired product were pooled 246 nm (e 6300); UV ,A, (pH 7) 246 nm (c 7400); UV A,, (pH and lyophilized until free of NH4HCOB.The residue was dissolved 11) 241 nm (c 15500); 'H NMR (Me2SO-d6)6 3.63 (d, 6, J H=, ~ in water (25 mL) and passed through a column of Dowex-50 H+ 10.85, 2 CH3), 4.76 (d, 1,J = 5.43, CitH), 5.05 (9, 2, CH2), 6.80, resin (50-100 mesh, 2 X 20 cm). The eluate was lyophilized to 7.18 (2 s, 2, CONH,), 7.40 (m, 5, CH&,&), 13.12 (d, 1,J",p = an amorphous powder to give 0.50 g (90.6%) of 23a; mp 160 "C 11.01 Hz), and other sugar protons. Anal. (C18H24N309P"20) 1660 (C==O),3170-3420 dec; IR (Kl3r) v 1075 (COC), 1230 (M), C, H, N, P. (pH 4-Hydroxy-3-~-~-ribofuranosylpyrazole-5-carboxam~de(NH,, OH) cm-'; UV ,A (pH 1)257 nm (e 15900); UV ,A, 7 and 11) 257 nm (c 15700); 'H NMR (Me2SO-d6)6 4.70 (d, 1, 5'-(Di-O-methyl phosphate) (20b). In the same manner as for J = 6.72 Hz, C1rH of 17), 5.03 (9, 2, CH2), 5.93 (d, 1, J = 5.19 Hz, 18, hydrogenation of 20a (2.0 g) in MeOH (100 mL) containing C1,H of Ado), 7.40 (m, 9, (345, CONH2, and NH,), 8.16, 8.48 (2 formic acid (5 mL) in the presence of 20% Pd/C (150 mg) gave s, 2, C2H and C8H of Ado), and other sugar protons. Anal. the title compound, which was purified on a silica gel column (2 X 40 cm) using CHC13/MeOH (6:1, v/v) as the eluent. Crys(C2&32N80~5P2*2H20)C, H, N, P. 5 -Carbam o y 1- 3- 8-D-ri bo f u ra no 8 y 1 p y r a z o 1y 1 ( 5-5') tallization from aqueous ethanol gave an analytical sample, 1.25 adenosine Pyrophosphate (PAD, 23b). To a solution of 23a g (75%); mp 178-180 "C; IR (KBr) v 1040 (POC), 1680 (C=O), (100 mg) in 95% ethanol (25 mL) was added 20% Pd(OH),/C 2940-3250 (OH, NH2) cm-'; UV A,, (pH 1) 262.5 nm (e 6500); (25 mg) and cyclohexene (8 mL), and the mixture was heated (pH 7) 265.5 nm (c 4700), 305 (2900); UV ,A, (pH 11) UV ,A, under reflux for 2 days. The catalyst was removed by filtration 307 nm (e 8900); 'H NMR (Me2SO-d6)6 3.64 (d, 6, J H=, ~ 11.81, on a Celite pad and the fiitrate evaporated to dryness. The residue 2 OCH3), 4.78 (d, 1, J = 5.08 Hz, CltH), 7.44 (br s, 2, CONH2), was dissolved in water (15 mL), adjusted to pH 7 with NH4HC03, 8.83 (br s, 1,C4-OH),12.92 (br s, 1,NH), and other sugar protons. and applied to a DEAE-Sephadex column (2.5 X 45 cm) equiliAnal. (C11H18N309P-'/2H20) C, H, N, P. 4-Hydroxy-3-~-~-ribofuranosylpyrazole-5-carboxamide brated with 0.05 M NH4HCOB.The column was washed with 0.05 M NaHC03 (150 mL) and eluted with a gradient of 0.05 M 3'$- (Cyc1ic)phosphate (Pyrazofurin 3',5'- (Cyclic)phosphate, NH4HC03 (200 mL) to 5 N NH4HC03 (200 mL) at a flow rate 21b). Compound 17 (0.50 g, 1.05 mmol) was dried by repeated of 3 mL/min. Fractions containing the desired product were evaporation with anhydrous pyridine. The residual syrup was pooled and lyophilized until free from NH4HCOB. The aqueous dissolved in dry pyridine (15 mL) and added dropwise to a resolution (25 mL) of the residue was passed through a Dowex-50 fluxing dioxane (200 mL) solution of dicyclohexylcarbodiimide H+ resin column (2 X 25 cm) and the eluate was lyophilized (DCC; 1.24 g, 6 mmol). After all the pyridine was added, the repeatedly to yield PAD, 85 mg (93%);mp 175 "C dec; IR (KBr) solution was refluxed an additional 6 h. The reaction mixture v 1060 (POC), 1230 (P=O), 1640 (CEO), 3000-3400 ("2, OH) was evaporated to dryness, and the residue was dissolved in water (pH 7) 259 nm cm-'; UV,,A (pH 1)259 nm (e 17000); UV ,A, (20 mL) and extracted with ether (3 X 50 mL). The aqueous (e 18000);UV ,A, (pH 11)259 nm (c 15400),306 (8700); 'H NMR solution was adjusted to pH 7 with NH4HC03and applied to a (Me2SO-d6)6 4.72 (d, 1,J = 6.10 Hz, C1,H of 171, 5.92 (d, 1,J = DEAE-Sephadex column (25 X 45 cm) equilibrated with 0.05 M 4.06 Hz, C1,H of Ado), 7.28, 7.41 (2 s, 4, CONH2 and NH2),8.15, NH4HCOB. The column was washed with 0.05 M NH4HC03(500 8.49 (2 s, 2, C8H and C2H of Ado), and other sugar protons. Anal. mL) at a flow rate of 3 mL/min. Fractions containing the cyclic phosphate were pooled and lyophilized until free of NH4HC03. (C19H26N8015P2*3H20)C, H, N, P. Antiviral Evaluation. Test compounds were evaluated for The residue was then dissolved in water (15 mL) and passed their ability to inhibit virus-induced cytopathic effect (CPE) through a column of Dowex-50 H+ resin (50-100 mesh, 2 X 15 produced by herpes simplex virus type 2 (HSV-2, 333), vaccinia cm). The eluate was lyophilized to a white powder (21a, 0.35 g), virus (VV), measles, and parainfluenza virus type 3 (para-3) in which was used for debenzylation studies without further puriAfrican green monkey kidney (Vero) cells (American Type Culture fication. Collection, Rockville, MD). Vero cells were maintained in anA solution of 21a (0.10 g) in 95% EtOH (25 mL) containing tibiotic-free Eagle minimum essential medium (EMEM) with 20% Pd(OH)2/C (25 mg) and cyclohexene (8 mL) was heated Earle's salts supplemented with 10% heat-inactivated newborn under reflux for 2 h. The catalyst was removed by filtration on bovine serum (Grand Island Biological Co., Grand Island, NY). a Celite pad and the filtrate evaporated to dryness. The residue For antiviral experiments, cells were inoculated into 96-well tissue was dissolved in water (25 mL), adjusted to pH 7 with NH4HC03, culture plates (Corning Glassworks, Corning, NY) at a concenand applied to a DEAE-Sephadex column (1X 15 cm) equilibrated tration of 4 X lo4 cells/0.2 mL per well and cultured for 24 h at with 0.05 M NH4HC03. The column was washed with 0.05 M 37 "C in 5% C02 to confluency. NH4HC0, (200 mL) and then eluted with a gradient of 0.05 M Monolayers were inoculated with a predetermined number of NH4HC03(250 mL) to 1.0 M NH4HC03(250 mL) at a flow rate TCIDw (50% tissue culture infective dose) units of virus that will of 3 mL/min. Fractions containing the desired product were produce complete destruction of the cell monolayer in 72 h. The pooled and lyophilized until free of NH4HC03. The residue was number of TCID,, units in 0.1 mL/well were as follows: HSV-2, dissolved in water (20 mL) and passed through a Dowex-50 Ht 100; VV, 200; measles, 100; para-3,60. After 30-min adsorption resin column (1 X 15 cm). The eluate was lyophilized to give a at 37 "C, test compounds were added (0.1 mL/well) in seven 0.5 light tan colored powder, which was triturated with anhydrous to 1 X M resulting log concentrations ranging from 1 X MeOH, filtered, and dried to yield 78 mg of pure 21b; mp 133 in final well concentrations of 5 X lo4 to 5 x M. Active "C dec; IR (KBr) v 1060 (POC), 1220 (P=O), 1650 (C=O), compounds were tested at concentrations of 5 X to 5 X 3000-3440 (NH2,OH) cm-'; UV ,A, (pH 1) 263 nm (c 3700);UV M. At each concentration, duplicate wells were used for evaluation ,A, (pH 7) 268 nm (e 2800); UV ,A, (pH 11)235 nm ( e 2900), of antiviral activity and single uninfected wells for cytotoxicity 305 (5700); 'H NMR (Me2SO-d6)6 4.89 (s, 1, C1,H), 7.43 (br s, evaluation. 3, C4-OH, CONH2),and other sugar protons. Anal. (C9H12N3OBP*~H,O) C, H, N, P. After 72-h incubation at 37 "C in 5% C02/air, the concentration 4-(Benzyloxy)-5-carbamoyl-3-~-~-ribofuranosyl- of compound that produced 50% inhibition of viral CPE was pyrazolyl(5-+5')adenosine Pyrophosphate (23a). A mixture determined (ED5& A given concentration of compound was of 17 (0.30 g, 0.63 mmol) and adenosine 5'-phosphoric di-n-buconsidered cytotoxic if it produced any microscopically visible tylphosphinothioic anhydride46(22;0.62 g, 1.26 mmol) was renchanges in cellular morphology or in the density of the cell dered anhydrous by repeated evaporation with dry pyridine. The monolayer due to lysis, rounding up, or detachment of cells. The residue was dissolved in dry pyridine (50 mL) to which was added lowest concentration where cytotoxicity to uninfected, confluent Vero cell monolayers was observed was considered to be the AgN03 (0.68 g, 4 mmol) and the mixture was stirred overnight maximum tolerated concentration (MTC). with the exclusion of moisture. H2Swas bubbled into the reaction mixture for 15 min, and the black precipitate that formed was Cytostatic Activity Evaluation. A. L1210 and P388 Leuremoved by filtration. The filtrate was evaporated to dryness. kemias. Compounds were evaluated for their ability to inhibit The aqueous solution of the residue was adjusted to pH 7 with growth of murine leukemia L1210 and lymphoid neoplasm P388

278

J . Med. Chem. 1986,29, 278-286

(American Type Culture Collection, Rockville, MD) maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Grand Island Biological Co., Grand Island, NY) and 20 mM Hepes buffer. For growth experiments, cells were adjusted to 1 X 105cells/mL and distributed to 24-well tissue culture plates (0.5 mL/well). Test compounds were dissolved in growth medium, sterilized by passage through an 0.22-pm membrane filter, serially diluted, and added to w e b (0.5mL/well). Compounds were tested in duplicate at log concentrations ranging from 1 X to 1 X M. Following 48-h incubation at 37 “C, cell counts were determined with a Coulter Model ZF cell counter. Cell growth in the presence of test compounds was expressed as a percentage of growth in untreated control wells and the concentration of compound producing 50% inhibition of cell growth was determined (ID,,,). B. B16 Melanoma and L e w i s Lung Carcinoma. Lewis Lung carcinoma was maintained as a monolayer in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum. B16 melanoma was maintained as a monolayer in Eagle’s minimum essential medium (MEM) containing 10% heat-inactivated fetal calf serum, 0.1 mM MEM nonessential amino acids, 1mM sodium pyruvate, and MEM vitamin solution. For determination of cell growth inhibition using either cell line, cells were seeded at 2.5 X lo4 cells per well in 24 tissue culture plates. Cells were grown 24 h at 37 “C in 5% COB,and then growth medium was replaced with medium containing the compound of interest at log concentrations ranging from 1 x to 1 X M. After an

additional 72-h incubation, cells were washed twice with phosphate buffered saline, trypsized to single cell suspensions,and counted with a Coulter Model ZF cell counter. Cell growth at each dose level was expressed as a percentage of growth in control wells and the dose resulting 50% inhibition of growth was determined. Acknowledgment. This work was supported in p a r t by Contract DAMD-17-79-C-9046 with t h e U S . Army Medical Research a n d Development Command, Washington, DC. This is Contribution No. 5003 t o the USAMRIID Drug Development Program. We t h a n k Ratan B. Bhappu for large-scale syntheses of certain chemical intermediates. Registry No. 1, 30868-30-5; 4,99298-13-2;4 (tetraacetyl derivative), 99298-21-2;4 (5-carbonitrile),99298-22-3;5,99298-15-4; 6, 99298-16-5; 7, 41329-11-7; 8, 99298-14-3; 9, 59252-13-0; 9 (5carbonitrile), 99298-17-6; 10, 99298-18-7; 11, 99298-19-8; 12, 99298-20-1; 13, 99298-23-4; 14, 99298-24-5; 15, 99298-25-6; 16, 99298-22-3; 17, 99298-26-7; 18, 65446-02-8; 19a, 99298-27-8; 19b, 99298289; 20a, 99298-29-0; 20b, 99298-30-3;21a, 99298-31-4; 21b, 99298-32-5; 22, 57816-25-8; 23a, 99298-33-6; 23b, 99298-34-7. Supplementary Material Available: Tables of positional parameters (Tables V-VIII), anisotropic thermal parameters of the non-hydrogen atoms (Tables IX-XII), and positional and isotropic thermal parameters of the hydrogen atoms (Tables XIII-XVI) for compounds 1,8,7, and 20a, respectively (12 pages). Ordering information is given on any current masthead page.

Mapping the Turkey Erythrocyte p Receptor: A Distance Geometry Approach Marcel R. Linschoten, Teake Bultsma,* Ad P. IJzerman, a n d Hendrik Timmerman Department of Pharmacochemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Received September 18, 1984

Extensions and refinements of the receptor mapping method as originally developed by Crippen are presented. In a set of newly developed algorithms measures are taken to reduce the number of required energy parameters to a statistically acceptable degree. The most important measure is the incorporation of lipophilicity as a hydrophobic bonding parameter to describe the binding of parts of the ligands to lipophilic areas on the receptor. In order to test the applicability of our set of programs, we mapped the turkey erythrocyte p receptor using a data set of Bilezikian. It was found that the experimentally determined free energies of binding can be reasonably described using a nine-point geometrical representation of the receptor site and only six energy parameters. The deduced model predicts that the phenyl rings of phenylethanolamines and phenoxypropanolamines occupy different parts of the receptor site. Conformational flexibility of ligands poses a major problem in pharmacophoric pattern search a n d receptor mapping techniques. It has become clear t h a t i t is unrealistic to assume that ligands always bind t o the receptor in their conformations of minimal energy as the free energy of association generally outweighs t h e energy of a conformational change of the ligand. It is therefore necessary t o take into account all conformations having energies u p t o a few kilocalories/mole above t h e global minimum. Conformational flexibility can be conveniently condensed in a distance matrix. Considering t h e total set of possible conformations, the entries in t h e upper triangle of such a matrix usually are t h e maximum distances between any pair of preselected atoms or d u m m y points in t h e molecule, whereas the entries in the lower triangle are t h e corresponding minimum distances. T h e methodology of distance geometry is a powerful tool in handling t h e information contained in such matrices. During t h e last few years especially Crippenl has applied

(1) Crippen, G. J . Med. Chem. 1980, 23, 599.

t h e distance geometry approach as a receptor mapping technique. By comparing the distance matrices of the ligands in the d a t a set, Crippen was able t o deduce t h e common structural features of a set of ligands. Substituent points were subsequently positioned relative t o this common base group. Complementary receptor “site points” were then proposed t o account for t h e binding of t h e structural features of t h e ligand (“ligand points”). Furthermore, Crippen supposed t h a t the total binding energy of a ligand t o its receptor is equal t o t h e sum of t h e individual interactions between ligand points a n d site points. Given t h e experimentally determined free energy of binding, the method enables one t o calculate t h e individual binding energy contributions of any ligand point-site point interaction. Thus, next to t h e incorporation of conformational flexibility in t h e calculations, t h e strong point of t h e method is t h a t it enables t h e researcher t o propose new, stronger binding ligands on basis of t h e geometry a n d energy parameters of t h e deduced receptor site model. A major problem with respect t o t h e general applica-

0022-2623/8S/l829-0278$01.50/0 0 1986 American Chemical Society

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